Evanescent scattering imaging of single protein binding kinetics and DNA conformation changes

Evanescent illumination has been widely used to detect single biological macromolecules because it can notably enhance light-analyte interaction. However, the current evanescent single-molecule detection system usually requires specially designed microspheres or nanomaterials. Here we show that single protein detection and imaging can be realized on a plain glass surface by imaging the interference between the evanescent lights scattered by the single proteins and by the natural roughness of the cover glass. This allows us to quantify the sizes of single proteins, characterize the protein–antibody interactions at the single-molecule level, and analyze the heterogeneity of single protein binding behaviors. In addition, owing to the exponential distribution of evanescent field intensity, the evanescent imaging system can track the analyte axial movement with high resolution, which can be used to analyze the DNA conformation changes, providing one solution for detecting small molecules, such as microRNA. This work demonstrates a label-free single protein imaging method with ordinary consumables and may pave a road for detecting small biological molecules.

a-e, ESM images and image intensity histograms of 27.9 nm (a), 41.3 nm (b), 61.6 nm (c), 93.7nm (d), and 143.6 nm (e) polystyrene nanoparticles (PSNP), where the solid red curves are Gaussian fittings. Each measurement was repeated on three different cover glasses. Incident intensity: 2 kW cm -2 . Camera exposure time: 5 ms for 27.9 nm, 2 ms for 41.3 nm and 61.6 nm, 1 ms for 93.7 nm, 0.2 ms for 143.6 nm. The image intensity was normalized with an incident intensity of 60 kW cm -2 and a camera exposure time of 5 ms. The arrows indicate the position where the nanoparticle binds. The histogram width mainly results from the nanoparticle heterogeneity. The dynamic light scattering results show that the 27.9 nm, 41.3 nm, 61.6 nm, 93.7nm, and 143.6 nm PSNP have the diameter distribution width of 5.4 nm, 8.1 nm, 12.8 nm, 15.4 nm, and 15.6 nm, respectively. The experiments were repeated three times with similar results. f, ESM image intensity versus particle diameter. Horizontal lines represented the mean values, and the error bars were small, indicating good ESM measurement reproducibility. The effective particle diameter as discussed in Supplementary Note 1 was employed for the fitting. The cross sign indicates the image intensity of the PSNP with an effective diameter of 100 nm, which is used for studying the relationship between image intensity and incident wavelength shown in Figure 1g. The sample sizes are presented in a-e, and the counts come from independent nanoparticles. The centers and error bars represent the mean values and standard deviations achieved from the image intensities determined by three independent measurements.
Supplementary Figure 6 Compare the light induced heating effect between PSM and ESM by monitoring the temperatureresponsive phase transition of polymer.
Some polymer solutions will produce large vesicles when the solution temperature is higher than the phase transition temperature (J. Phys. Chem. B 2007, 111, 6, 1262-1270. The 0.236 g/L polyethylene oxide (PEO) dissolved in 300 mM NaH 2 PO 4 and 240 mM NaH 2 PO 4 aqueous solutions have the phase transition temperature at 62 °C and 66 °C, respectively. Phase transition was observed when flowing PEO in 300 mM NaH 2 PO 4 solution onto the gold film surface of PSM (Supplementary Figure 6a), and not observed when flowing PEO in 240 mM NaH 2 PO 4 solution (Supplementary Figure 6b). This indicates the PSM sensor surface temperature is between 62°C and 66 °C under the incident intensity of 4 kW cm -2 . This is close to the upper temperature limit for protein detection (Journal of Pharmaceutical and Biomedical Analysis 2020, 189, 113399), so it is hard to increase the incident intensity further. In contrast, phase transition was not observed on the glass surface of ESM under the incident intensity of 60 kW cm -2 when flowing 1 g/L cellulose in 300mM Na 2 HPO 4 aqueous solution with a phase transition temperature of 33 °C. This indicates a much smaller heating effect on the glass surface than the gold surface, thus allowing the ESM to employ the incident intensity of 60 kW/cm 2 , which is ~20 times higher incident intensity than reported value for PSM (Nat Methods 17, 1010-1017(2020). Camera exposure time is 0.1 ms for PSM, and 5 ms for ESM. A detailed study of the heating effect will be published in an upcoming article.

Supplementary Figure 7
Simulated surface plasmon resonance (SPR) curve for the gold-water interface under different incident wavelengths.
Winspall data analysis software (http://res-tec.de/downloads.html) is used to simulate SPR curves under different wavelengths. The gold refractive index is 1.52 + 1.96i, 0.48 + 2.36i, 0.24 + 3.23i, 0.19+ 3.59i, and 0.17+3.87i under incident wavelengths of 450 nm, 532 nm, 600 nm, 633 nm, and 660 nm, respectively. BK7 cover glass has a refractive index of ~1.52 under visible light (refractiveindex.info). Gold film thickness is set at 50 nm. The simulation shows that the SPR cannot be observed with blue and green incident light, and the SPR angle is ~75° under the incident wavelength of 600 nm. The commonly used immersion oil objective has a numerical aperture of 1.49, corresponding to the largest incident angle of ~78.6°. Thus, the SPR condition is hard to be achieved with an incident wavelength of 600 nm to construct PSM, and PSM usually requires the incident wavelength longer than 600 nm. In contrast, the BK7 cover glass and water have a stable refractive index over the entire visible light range, so ESM allows a wide incident wavelength range.

Supplementary Figure 8
ESM image intensity versus particle diameter under the incident wavelengths of 405 nm (a), 488 nm (b), 532 nm (c), and 660 nm (d). 27.9 nm, 41.3 nm, 61.6 nm, 93.7nm, and 143.6 nm polystyrene nanoparticles were measured on three different cover glasses. Horizontal lines represented the mean values, and the error bars were small, indicating good ESM measurement reproducibility. The effective particle diameter as discussed in Supplementary Note 1 was employed for the fitting. Incident intensity: 2 kW cm -2 . The image intensity was normalized with an incident intensity of 60 kW cm -2 and a camera exposure time of 5 ms. The cross sign indicates the image intensity of the PSNP with an effective diameter of 100 nm, which is used for studying the relationship between image intensity and incident wavelength shown in Figure 1g. The sample sizes are presented in the figures, and the counts come from independent nanoparticles. The centers and error bars represent the mean values and standard deviations achieved from the image intensities determined by three independent measurements.

Supplementary Figure 9
Stability of the surface chemistry under the illumination of high intensity 405 nm and 450 nm light.
The cover glass was modified with NHS groups (Methods), and high concentration protein and 26 nm polystyrene nanoparticle solutions were flowed onto the sensor surface for binding. Then, the cover glass was illuminated by the 405 nm (a), and 450 nm (b) laser with an incident intensity of 60 kW cm -2 . For the incident wavelength of 405 nm, the analytes will leave the surface within about 5 minutes (300 s), whereas the surface chemical state is stable under the incident wavelength of 450 nm. This is likely due to the high photon energy of the near UV light (405 nm) damaging the chemical linkers and detaching the analyte from the sensor surface. Camera exposure time: 0.5 ms for the incident wavelength of 405 nm, and 2.5 ms for the incident wavelength of 450 nm. The solid red curves are Gaussian fitting. The arrows indicate the minor peaks shown at the position with ~ two times higher intensity than the mean values. The peak height does not scale with the protein concentration, and becomes a tail at a high analyte concentration, indicating that they are not created by dimmers. The possible reason is that two or more molecules were simultaneously falling within the same Airy disk area with a diameter of ~ 1 µm, which cannot be resolved in the image and counted as a single binding event. Incident wavelength: 450 nm. Incident intensity: 60 kW cm -2 . Camera exposure time: 5 ms. Figure 12 ESM image intensity histograms measuring BSA proteins in different regions.

Supplementary
The BSA proteins recognized in the central area present slightly higher image intensity than those in the marginal area, and thus the proteins binding on the central illumination area will be recognized more easily. Incident wavelength: 450 nm. Incident intensity: 60 kW cm -2 . Camera exposure time: 5 ms.  (a) ESM images and intensity histograms of gold nanoparticles with different diameters at the incident wavelength of 532 nm. The image intensity was normalized with the incident intensity of 2 kW cm -2 and the camera exposure time of 1.5 ms. The experiments were repeated three times with similar results. (b) ESM image intensity versus gold nanoparticle diameter, where the z-distance dependence of evanescent wave is considered (Supplementary Note 1). The image intensity follows a power law of d 5.5 , where the exponent is close to six, indicating that the nanoparticle scattering dominates the image intensity.

Supplementary Figure 16
Effective spring constant statistical distribution of gold nanoparticles linked by ssDNA molecules measured on three cover glasses before and after hybridization with miRNA, where the solid curves are Gaussian fitting. Glass No. 1 is shown in Figure 4i. Incident wavelength is 532 nm, and incident light intensity and camera exposure time are 2 kW cm -2 and 0.2 ms.

Supplementary Note 1 Effective diameter correction
The evanescent field decreases exponentially from the surface (z-direction) into the solution. In other words, the scattering of the evanescent field by a finite-size object depends on the distance (z) from the surface (Supplementary Figure 17). The effective scattering diameter D eff and volume V eff of the analyte can be given by where z is the distance from the gold surface, R is the radius of the analyte, D is diameter of analyte, and l is the decay length of the evanescent field (PNAS, 2010, 107(37): 16028-16032).
Supplementary Figure 17 Effective diameter correction model.
The decay length l of the evanescent field can be calculated by where λ is the incident wavelength, θ is the incident angle, n glass and n solution are the refractive indices of cover glass and solution, respectively (https://www.olympus-lifescience.com/en/microscoperesource/primer/java/tirf/penetration/). This study sets the incident angle at 65 o . n glass and n solution are 1.52 for BK7 cover glass and 1.33 for water, respectively. For an incident wavelength of 405 nm, 450 nm, 488 nm, 532 nm, and 660 nm, the decay length l of the evanescent field can be estimated to be 90 nm, 100 nm, 108 nm, 118 nm, and 146 nm, respectively.

Estimation with Rayleigh scattering model
The evanescent wave is one kind of subwavelength optical wave, owning more complex propagation and scattering properties than the far-field light (Nature Nanotech. 7, 668-672 (2012); Angew. Chem. Int. Ed. 58, 572 -576 (2019)). This complex behavior usually cannot be precisely described by current theory, while the numerical iteration and theoretical approximation analysis have shown that the object has larger scattering cross-section in the evanescent field than the far-field light field (Surface Science, 590, 173-180 (2005); Phys. Rev. B 92, 245419 ( 2015); Nat Commun 11, 4768 (2020)). Nevertheless, as a rough estimation, Rayleigh scattering model can be used to describe the evanescent scattering behaviors of small objects (Anal. Chem. 86, 8992-8997 (2014)).
Textbook shows that the Rayleigh scattering cross-section σ can be given by (https://en.wikipedia.org/wiki/Rayleigh_scattering) n a n m 2 -1 n a n m 2 +2 2 , N2.1 where d is the analyte diameter, λ is the incident wavelength, and n a and n m are the refractive index of analyte and media, respectively. The media is water in this study. The equation (N2.1) shows that the scattering intensity of nanoparticle scales with λ -4 , close to the experimental results shown in Figure 1g. The experimental results show that the polystyrene nanoparticles present ~24% higher image intensity than proteins with the same diameter.

Supplementary
After configuring the incident wavelength and media, the Rayleigh scattering model shows that the analyte diameter and refractive index determine the scattering intensity. The polystyrene has a refractive index of 1.61 (refractiveindex.info) under the incident wavelength of 450 nm. On the other hand, the proteins in the evanescent field are reported to have a refractive index of 1. 48 (Meas. Sci. Technol. 2006, 17, 932-938). Therefore, calculation with the Rayleigh scattering model shows that the protein scattering cross-section will equal ~30% of the scattering cross-section of polystyrene nanoparticles with the same diameter, leading to that the protein image intensity should be ~82% smaller than the image intensity of polystyrene nanoparticles with the same diameter under interference detection conditions. However, the experiments show that the ESM image intensities of proteins are only ~24% smaller than that of the polystyrene nanoparticles with the same diameter (Supplementary Figure 18). This discrepancy should be because of the different binding conditions. The polystyrene nanoparticles bind to the poly-l-lysine modified surface via electrostatic adsorption, and proteins bind to the carboxyl group modified surface via covalent bonding. It should be noted that the cubic power law of d 3 , where d is the object diameter, achieved in Figure 2 does not conflict with the results shown for iSCAT or mass photometry. As shown in Supplementary Figure 20, we can also find that the ESM image intensity is proportional to the mass. We use diameter here to easily compare the ESM measurement results with the analyte diameter determined by the dynamic light scattering. The Rayleigh scattering model can also be used to estimate the system signal-to-noise ratio (SNR) theoretically. For the ESM measurement of single proteins, the image intensity I was determined by the interference between evanescent wave scattered by the surface roughness E b and protein E s as

Supplementary
where θ is the phase difference between E b and E s .
In the shot noise dominant system, the SNR is determined by the shot noise, which can be shown as 3) The phase difference θ is about zero in ESM because of the short distance between scattering sites of surface roughness and analyte binding positions (ACS Photonics 8, 2227-2233 (2021)). The equation shows that the SNR is mainly determined by the scattering amplitude detected by the camera in the shot noise dominant system.
To estimate the theoretical SNR limit for the ESM system, the total Rayleigh scattering intensity I total of one protein molecule is firstly estimated by where n s and n m are the refractive indices of analyte and medium, λ is the incident wavelength, d is the analyte diameter, P is the incident light intensity, and t is the average period. For the ESM used in this study, n s = 1.48, n m = 1.33, λ = 450 nm, and t = 0.05 s. Considering the single photon energy of ~1.2398/(0.45 µm) eV and the 5 x intensity enhancement of evanescent field, the total scattering intensity of one object in the ESM system can be expressed as I total = 5 × 10 -4 × d nm 6 × P kWcm -2 photons, (N2.5) The objective collects the scattering photons in perpendicular to the propagation direction of the evanescent wave, and the collection efficiency can be calculated with the equation in a spherical coordinate system of where and are the polar angle and azimuthal angle, respectively. The objective collection angle for the ESM can be calculated by where NA is the objective numerical aperture, and n m = 1.33 is used to correct the effect of water refraction on the scattering light collection. For the objective with an NA of 0.42, the collection efficiency is calculated to be ~2.5 %.
For the detection of BSA with a diameter of 8.4 nm under the incident intensity of 60 kW cm -2 and the average period of 50 ms, the total number of scattering photons is ~10000 based on equation (N2.5), and the number of photons collected by the objective is ~250 based on equations (N2.6) and (N2.7). Thus, the theoretical limit of SNR is ~ 30 under perfect conditions based on equation (N2.3). The transmission ratio of the imaging objective and tube lens in the ESM is measured to be ~70%, and the quantum efficiency of the XIEMA MQ003 camera is ~40% for the incident wavelength of 450 nm, so ~70 photoelectrons actually contribute to the sensor output. Considering ~40% higher noise brought by the differential processing, the shot noise limited SNR of our setup for the BSA detection with ESM employing the incident intensity of 60 kW cm -2 and the average period of 50 ms is ~ 10, agreeing with the experimental results.

Supplementary Note 3 Image Intensity calculation
The object usually creates bright spots with more complex patterns than conventional Airy patterns on the evanescent scattering images, which may be caused by the delocalization features of evanescent waves along the surface (Nat Methods 17, 1010-1017 (2020)). Supplementary Figure 21 shows that both ESM images of single 143.6 nm polystyrene nanoparticle (PSNP) and BSA protein have complex intensity distribution, especially at the marginal area. Therefore, we do not employ the two-dimensional Gaussian fitting to process the images but average the intensities of all pixels within the diffraction-limited area to produce the sensor output. The diameter of the diffraction-limited area was estimated to be ~1.07 µm by dividing the incident wavelength of 450 nm with the imaging objective numerical aperture of 0.42. The objective magnification is 50x, and the XIMEA MQ003 camera pixel size is 7.4 µm, so the diameter of a diffraction-limited area on the image can be estimated to be 1.07*50/7.4 ~ 7.2 pixels. To ensure taking all the pixels receiving the light into consideration, we employ the circle with a diameter of 8 pixels in the TrackMate v6.0.1 to measure the mean intensity of bright spots achieved by ESM. Supplementary Figure 21 shows that negative intensities exist within the diffraction-limited area. In addition, due to the finite size of camera pixels, the TrackMate v6.0.1 calculates the average value of intensities of 61 pixels with setting the circle diameter to be 8 pixels, where the total area should be closer to a square shape rather than a round shape, resulting in taking some blank pixels into the calculation. These two factors lead to the mean intensity being usually 5~7 smaller than the maximum intensity of the bright spots (Supplementary Figure 22). The ratio of maximum intensity to mean intensity fluctuates. This is likely because the maximum intensity is a single-pixel value, and is subject to shot noise and camera pixel noise, especially for the weak signals with low signal to noise ratios, such as the measurement results of 27.9 nm and 41.3 nm polystyrene nanoparticles with an incident intensity of 2 kW cm -2 and BSA proteins with an incident intensity of 60 kW cm -2 .

Supplementary Note 4 Tracking precision estimation
For single particle tracking, the tracking precision in one dimension can be theoretically described by where σ μi is the standard deviation of localization positions at i-th dimension, N is the photo number, a is the pixel size/magnification, s is the standard deviation of Gaussian distribution, and b is the standard deviation of camera output in the dark (Science, 300, 2061(Science, 300, -2065(Science, 300, (2003). The photon number N was used to estimate the shot noise limited signal to noise ratio (SNR), which is equal to the square root of photon number. For interference detection, the photon number N can be represented by the square of the shot noise limited SNR (Nat Commun 5, 4495 (2014)

Relationship between the ESM image intensity and z-displacement
The relation between evanescent field intensity I E and z-displacement can be given by (J. Am. Chem. Soc. 2019, 141, 40, 16071-16078) I E = I E0 e -z/l , N5.1 where I E0 is the evanescent field intensity at z = 0, and l is the decay length of the evanescent field, which is 100 nm, 118 nm for the incident wavelength of 450 nm, and 532 nm, respectively (Supplementary Note 1). Under pure scattering conditions, the z-displacement of the analyte can be estimated by Under interference conditions, the scattering amplitude E S of the analyte can be estimated by E s = I E = I E0 e -z/2l , N5.3 So, the z-displacement of the analyte can be estimated by The statistical distribution of fluctuating z-displacement amplitude can be used for evaluating the free energy profiles of one binding pair according to where A is a constant that can be determined by normalization of P(z), k B is the Boltzmann constant, and T is the temperature (PNAS 2003, 100 (20), 11378-11381;PNAS 2020, 117 (44). 27148-27153). The effective spring constant k f can be determined from the simplified expression of free energy profile near equilibrium shown as where G(0) is the free energy at equilibrium (J. Am. Chem. Soc. 2019, 141, 40, 16071-16078).
For the interference measurement, the background intensity fluctuation in the region adjacent to the protein binding site was calculated (Supplementary Figure 23) and shows much smaller fluctuations, indicating that the IgM binding dominated this z-axis movement shown in Figure 4b.
For the gold nanoparticle tracking, the nanoparticle intensity is independent of background intensity, thus allowing higher tracking precision. To evaluate the tracking precision, the intensities of all nanoparticles recorded in each frame were averaged for averaging out the random thermal fluctuations. Although the average results should still contain some contributions from thermal fluctuations because the number of particles is finite, the fluctuation after average still reveals that the tracking precision is at least 0.04 nm.  where ρ is the fluid density of ~10 3 kg/m 3 , u is the mean flow rate of ~0.03 = 3× 10 -2 m/s in the experiment with a volume flow rate of 200 µL/min, μ is the dynamic viscosity of ~10 -3 Pa/s for PBS buffer (Nat. Chem. 13, 428-434 (2021)). Reynolds number is much smaller than 2300, indicating that the waterflow in our flow cell is laminar flow (https://en.wikipedia.org/wiki/Reynolds_number). The viscous damping force F d can be estimated with Stoke's equation of F d = 6πηr dz dt = 6 × π × 10 -3 × 20 × 10 -9 × 0.3 ~ 1 × 10 -9 1 × 10 -1 ≈ 1.1 ~ 3.7 × 10 -6 pN, N6.3 where η is solvent viscosity (~10 3 Pa·s for water), r is the nanoparticle radius, the measurement duration dt is ~0.1 s for the vertical tracking of nanoparticle, and dz is the nanoparticle movement distance within the measurement duration, which is ~0.3 nm, and 1 nm for nanoparticles on hybridized duplex structure, and ssDNA, respectively where b is the Kuhn length in nanometer, n is the number of segments with a length of b, 0.4 nm is the length of one base pair, 26 is the number of base pairs of the DNA used in this experiment (J. Chem. Phys. 142, 194902 (2015); Nat Commun 11, 4768 (2020)). ssDNA has the Kuhn length ranging from ~1.5 nm to ~8 nm (Science 271 (5250), 795-799 (1996); Macromolecules 30, 5763-5765 (1997)), and rigid dsDNA has Kuhn length of ~70 nm (J. Chem. Phys. 130, 215105 (2009)). The entropy force of fully stretched ssDNA linker before and after hybridization with microRNA can be estimated to be 0.1 to 15 pN and 10 -4 pN, respectively.
The restoring force F f of DNA linked nanoparticles can be estimated by F f = k f z = 0.19 × 1 × 10 -9~ 0.36 × 0.3 × 10 -9 ≈ 190 ~ 108 pN, N6.5 where k f is the effective spring constant (Figure 4i), and z is nanoparticle movement distance (Figure 4e). These results clearly show that the restoring force resulting from the DNA linker is much larger than the viscous damping force and entropy force and should dominate the nanoparticle motions.